Parkinson's disease is a neurodegenerative disorder that results from significant and selective loss of neurons in targeted brain regions, specifically dopaminergic neurons within in the brain region Substantia nigra. Neurodegeneration in PD results in locomotion defects such as bradykinesia, dyskinesia, and resting tremors. Although advances in identifying genetic risk factors for the neuronal pathology of the disease are being made, a complete picture of the cellular networks coupling these genetic abnormalities to neurotoxicity and neurodegenerative disease pathology remains a mystery. The long-term goal of this R21 proposal is to interrogate the effects of the small molecule NAB2 on the activity and specificity of the E3 ubiquitin ligase Nedd4 and to illuminate the molecular basis behind NAB2 alleviation of ?-synuclein-associated toxicity. In Aim 1 we will examine the ability of the Nedd4 mechanism and kinetics to be altered by chemical modulation will be interrogated through parallel computational, biophysical, and biochemical experiments. The binding mode of NAB2 will be validated using hydrogen-deuterium exchange mass spectrometry, revealing how the ligand engages its target. The biophysical characterization of NAB2 binding will be complimented by a series of mechanistic, in vitro assays which will interrogate Nedd4 function including single- and multi-turnover kinetics, alleviation of autoinhibition, and determination of mechanism (i.e. processive vs. distributive ubiquitylation). The proposed experiments will employ a set of semi-synthetic chemical probes to allow for direct assay of Nedd4 activity. Together, these experiments will determine the mode of action of NAB2 at an enzymatic level and will demonstrate how a small molecule can tune the enzymatic activity of Nedd4, further unraveling the nuances of its mechanism potential for regulation by small molecules. In Aim 2 we will examine the role of Nedd4 in the ?- synuclein toxicity network, we will conduct parallel bioinformatic and proximity-driven proteomics analyses to characterize the Nedd4 interactome. Bioinformatic tools will be employed to annotate characteristic structural motifs in the known Nedd4 interactome and ?-synuclein toxicity network, determining the predictability of Nedd4 substrates based on structural analyses and identifying putative Nedd4 substrates in the ?-synuclein toxicity network. This will be complemented by proximity-driven proteomics using the ascorbate peroxidase (APEX) mass spectrometry platform. In a relevant cellular model, Nedd4 will be expressed as a fusion with APEX, allowing for in situ biotinylation of interacting proteins. Subsequent streptavidin-based purification and protease digest coupled mass spectrometry will allow for characterization of the Nedd4 interactome. This model can be used for analysis +/- NAB2 and +/- toxicity, allowing for differential analysis of the Nedd4 interactome in response to chemical and cellular stimuli. Together, these experiments provide insight into the tunability of Nedd4 as an agent in the rescue of cellular toxicity.